Construction method of chondroitin-4-O-sulfotransferase mutant

By performing specific site mutations and designing fusion tags for C4ST, the problems of insufficient solubility and catalytic activity of C4ST were solved, and the industrial production of chondroitin sulfate A with high efficiency was realized.

CN122445602APending Publication Date: 2026-07-24JIANGNAN UNIV
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Patent Information

Application Number
CN202610593984.1
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-04-30
Publication Date
2026-07-24

AI Technical Summary

Technical Problem

Wild-type C4ST has low soluble expression levels and insufficient intrinsic specific enzyme activity, making it difficult to meet the requirements for industrial production of chondroitin sulfate A.

Method used

By making specific site mutations on the wild-type C4ST amino acid sequence, combining the fusion solubilizing tag SUMO and the linker peptide AE, a C4ST mutant was constructed and expressed in Escherichia coli or Pichia pastoris. The reaction conditions were optimized to improve enzyme activity.

Benefits of technology

The soluble expression level and catalytic activity of the C4ST mutant were significantly improved. The enzyme activity of the single-point mutant in Pichia pastoris shake flask reached 2840 U/L, and the enzyme activity of the three-point combination mutant in a 5 L fermenter reached 76328 U/L. The degree of sulfonation can reach 97.0%, which meets the requirements of industrial production.

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Abstract

The invention discloses a construction method of a chondroitin-4-O-sulfotransferase mutant, and belongs to the technical field of biological engineering. According to the invention, a far-end region of wild-type chondroitin-4-O-sulfotransferase (C4ST) is precisely modified through a semi-rational design guided by calculation, an SUMO tag and an AE connecting peptide are fused at an N end to improve soluble expression, and on this basis, a three-point combined mutant V74L / M117T / Y235V is constructed through a site-directed mutagenesis technology, the enzyme activity of the mutant in a 5L fermentation tank is as high as 76328 U / L, the specific enzyme activity is 327 U / mg, and the specific enzyme activity of the mutant in a 5L fermentation tank is as high as 76328 U / L; and the results are respectively 16.6 times and 1.3 times of those of a wild type. When the mutant is used for catalytic preparation of chondroitin sulfate A, the sulfonation degree can reach 97.0% after reaction is carried out at 37 DEG C for 48 h. According to the method, the catalytic efficiency of C4ST is remarkably improved, and an efficient and stable key technology is provided for efficient preparation of CSA through an enzyme method.
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